REGULATION OF SMOOTH MUSCLE CONTRACTILE PROPERTIES

平滑肌的收缩特性大致分为相性(快)和紧张性(慢)。时相肌的特点是力激活、力松弛和Vmax较快，而紧张性平滑肌的特点是力激活、力松弛和Vmax较慢。然而，调控平滑肌收缩特性的分子机制尚不清楚。这项资助的总体目标是确定平滑肌收缩特性的分子机制，并阐明快收缩和慢收缩特性的机制。我们的假设是，收缩蛋白的剪接异构体决定了平滑肌的收缩特性。检验这一假说的具体目的是确定MLC17(特异靶1)、MHC(特异靶2)和MLC磷酸酶(特异靶3)的剪接变异体是否是决定平滑肌收缩特性的分子决定因素。我们还将确定MLC磷酸酶的剪接变体是否影响激动剂诱导的力量增强的幅度或敏感性(特定目标3)。为了测试这些特定的目的，我们将强制在培养的胚胎主动脉和肌肌细胞中表达MLC17、MHC和MLC磷酸酶的两种剪接变体异构体。我们将确定在培养的主动脉和肌肌细胞中正常表达或不表达的异构体对培养的平滑肌细胞力学性能的影响。在强迫单个收缩蛋白表达后，我们将测定单个培养的平滑肌细胞的最大力、力激活和力松弛速率、Vmax和MLC20磷酸化，并将结果与未转染对照组的结果进行比较。这些实验将阐明单独表达一种收缩蛋白对培养的平滑肌细胞机械性能的影响。这些研究的结果将阐明决定平滑肌收缩特性的机制，并为未来研究疾病状态如何改变平滑肌的收缩特性奠定基础。

The contractile properties of smooth muscle are broadly classified as phasic (fast) and tonic (slow). Phasic smooth muscle is characterized by a rapid rates of force activation, force relaxation and Vmax, whereas tonic smooth muscle is characterized by slow rates of force activation, force relaxation and Vmax. However, the molecular mechanism that regulates the contractile properties of smooth muscle is unknown. The overall goal of this grant is to determine the molecular mechanism for the contractile properties of smooth muscle and to elucidate the mechanism for fast and slow contractile properties. Our hypothesis is that splice variant isoforms of contractile proteins determine the contractile properties of smooth muscle. The specific aims to test this hypothesis are to determine if splice variants of MLC17 (Specific Aim 1), MHC (Specific Aim 2) and MLC phosphatase (Specific Aim 3) are molecular determinants of the contractile properties of smooth muscle. We will also determine if splice variants isoforms of MLC phosphatase effect either the magnitude or sensitivity of agonist induced force enhancement (Specific Aim 3). To test these Specific Aims we will force the expression of both splice variant isoforms of MLC17, MHC and MLC phosphatase in cultured embryonic aortic and gizzard smooth muscle cells. We will determine the effects of the expression of the isoform normally present or not expressed in the cultured aortic and gizzard smooth muscle cells on the mechanical properties of cultured smooth muscle cells. After forcing the expression of a single contractile protein, we will determine the maximum force, the rates of force activation and force relaxation, Vmax, and MLC20 phosphorylation of single cultured smooth muscle cells and compare the results to those obtained in the non-transfected controls. These experiments will elucidate the effects of the expression of a single contractile protein, in isolation, on the mechanical properties of cultured smooth muscle cells. The results of these studies should elucidate the mechanism that determines the contractile properties of smooth muscle, and form a foundation for future investigation of how smooth muscle contractility is altered by disease states.